Many molecular biology applications, such as capillary electrophoresis, nucleotide sequencing, require the isolation of high quality nucleic acid preparations. Quality is a particularly important factor for capillary electrophoresis for all sequencing methods and for gene therapy protocols. Quantity is also an equally important consideration for some applications, for example, large scale genomic mapping and sequencing projects, which require the generation of hundreds of thousands of high quality DNA templates.
The emergence of new technologies such as Next Generation Sequencing (NGS) requires high quality DNA sample preparation with precise size control of certain groups of DNA fragments. After DNA fragmentation or shearing, the library construction process for next generation sequencing mostly requires fragments selection regardless of the platform. Obtaining high recovery post fragment selection is becoming an important contributor for the reduction of sequencing bias. For example, to prepare a DNA library for Illumina NGS platform, recovery of DNA fragments in the 150-500 bp range is critical for better sequencing results. Other NGS platforms require DNA fragments ranging between 300-700 bp.
There are two common methods to perform size selection for NGS library preparation. One method is to run the fragmented DNA into agarose gel and then cut the gel with selected DNA size and then recover the DNA fragments from gel. This method is accurate, but it is very slow and labor intensive.
Another common used method to prepare size selection of fragmented DNA is to bind DNA on magnetic beads coated with functional groups such as a carboxyl group by adjusting the concentration of polyethylene glycol (PEG) and salts. See U.S. Pat. No. 6,534,262. This method fragments DNA efficiently and can be easily adapted in an automated platform to process large number of samples at one time. However, this method does not work well for DNA fragmentation when the DNA fragments are larger than 400 bp. The unwanted large DNA fragments decrease the data quality for the subsequent NGS results and waste the capacity of the instrument. For an NGS platform that requires large DNA fragments, for instance the NGS platform for Roche 454 Genome Sequencer, this method is not suitable.
U.S. Pat. No. 5,234,809 describes a method that uses solid phase particles to specifically and/or non-specifically bind nucleic acids from a sample in the presence of chaotropic salts, such as guanidinium salt, sodium iodide, and potassium iodide. However, this method does not provide any size control on nucleic acids fragments.
Therefore, the growing application of next generation sequencing (NGS) demands new nucleic acid purification and fragment size selection methods that provide high nucleic acid recovery and precise fragment size control.